Nucleic acid sequence analysis is extremely important in many research, medical, and industrial fields. See, e.g., Caskey, Science 236:1223–1228 (1987); Landegren et al, Science 242:229–237 (1988); and Arnheim et al, Ann. Rev. Biochem. 61:131–156 (1992). The most commonly used sequence analysis technique is polymerase chain reaction (PCR). PCR and other sequence determination techniques involve extension of an oligonucleotide primer with a polymerase. Extension of a primer with a polymerase also occurs in vivo in DNA replication and in transcription of DNA to form RNA.
Fidelity of DNA replication in vivo is maintained, in part, by a 3′-to-5′ exonuclease proof-reading activity of the DNA polymerase. When an incorrect nucleotide is incorporated and forms a mismatch with the template, it is removed by the 3′-to-5′ exonuclease. The thermostable DNA polymerase most widely used for PCR, however, Thermus aquaticus (Taq) polymerase, lacks a 3′-to-5′ exonuclease.
Other methods of sequence determination or nucleic acid analysis involve ligation of oligonucleotides, or involve both ligation of oligonucleotides and polymerase extension of oligonucleotides. One technique is the oligonucleotide ligation assay (OLA) of Whiteley et al., U.S. Pat. No. 4,883,750. The method is used to determine the presence or absence of a target sequence in a sample of denatured template nucleic acid. Two oligonucleotide probes are designed so they will hybridize to the target sequence with the 5′ base of one oligonucleotide abutting the 3′ base of the other. If these two bases form perfect hybrds with the target sequence of the template DNA, then the oligonucleotides can be ligated together by DNA ligase. If the template DNA contains a mutation at one of those two bases in the target sequence, the oligonucleotides cannot be ligated. If a thermostable ligase is used, the reaction can be carried out for multiple cycles, just as in PCR. This can greatly improve the signal to noise ratio. (See Wu and Wallace, Genomics 4:560 (1989); Barany, Proc. Natl. Acad. Sci. USA 88:189(1991).) Assays that combine OLA and PCR are described in Eggerding, U.S. Pat. No. 6,130,073; and Nickerson et al., Proc. Natl. Acad. Sci. USA 87:8923–8927 (1990).
In PCR and other polymerase-based assays using oligonucleotides, as well as in ligation-based assays, unextended or unligated oligonucleotides often need to be removed from the reaction mixture for subsequent analysis steps. This is true, for instance, in nested PCR and sequencing of PCR products, or when the amplified product is to be hybridized to a sequence to which the primer would competitively hybridize. Hence, there is a need for techniques that quickly and easily remove unextended oligonucleotides from polymerase and ligase reaction mixtures.